School of Basic Medicine, Guangxi University of Traditional Chinese Medicine, Nanning 530200, China.
Guangxi Key Laboratory of Liver and Spleen Visceral Manifestations in Chinese Medicine, Guangxi University of Traditional Chinese Medicine, Nanning 530200, China.
Mar Drugs. 2024 Jul 27;22(8):345. doi: 10.3390/md22080345.
meat is the main by-product of the pearl harvesting industry. It is rich in nutrition, containing a lot of protein and peptides, and holds significant value for both medicine and food. In this study, a new active protein was discovered and expressed heterogeneously through bioinformatics analysis. It was then identified using Western blot, molecular weight, and mass spectrometry. The antibacterial activity, hemolysis activity, antioxidant activity, and Angiotensin-Converting Enzyme II (ACE2) inhibitory activity were investigated. An unknown functional protein was screened through the Uniprot protein database, and its primary structure did not resemble existing proteins. It was an α-helical cationic polypeptide we named PFAP-1. The codon-optimized full-length gene was synthesized and inserted into the prokaryotic expression vector pET-30a. The induced expression conditions were determined with a final isopropyl-β-d-thiogalactoside (IPTG) concentration of 0.2 mM, an induction temperature of 15 °C, and an induction time of 16 h. The recombinant PFAP-1 protein, with low endotoxin and sterility, was successfully prepared. The recombinant PFAP-1 protein exhibited strong antibacterial activity against methicillin-resistant (MRSA) in vitro, and the diameter of the inhibition zone was 15.99 ± 0.02 mm. Its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 37.5 μg/mL and 150 μg/mL, respectively, and its hemolytic activity was low (11.21%) at the bactericidal concentration. The recombinant PFAP-1 protein significantly inhibited the formation of MRSA biofilm and eradicated MRSA biofilm. It also demonstrated potent 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) scavenging activity with a half-maximal inhibitory concentration (IC) of 40.83 μg/mL. The IC of ACE2 inhibition was 5.66 μg/mL. Molecular docking results revealed that the optimal docking fraction of PFAP-1 protein and ACE2 protein was -267.78 kcal/mol, with a confidence level of 0.913. The stable binding complex was primarily formed through nine groups of hydrogen bonds, three groups of salt bridges, and numerous hydrophobic interactions. In conclusion, recombinant PFAP-1 can serve as a promising active protein in food, cosmetics, or medicine.
肉是珍珠养殖行业的主要副产品。它富含营养，含有大量的蛋白质和肽，对医药和食品都具有重要价值。在这项研究中，通过生物信息学分析发现并异源表达了一种新的活性蛋白。然后使用 Western blot、分子量和质谱对其进行鉴定。研究了其抗菌活性、溶血活性、抗氧化活性和血管紧张素转化酶 II（ACE2）抑制活性。通过 Uniprot 蛋白质数据库筛选出一种未知功能的蛋白质，其一级结构与现有蛋白质不相似。这是一种我们命名为 PFAP-1 的α-螺旋阳离子多肽。合成并插入原核表达载体 pET-30a 中的是经过密码子优化的全长基因。最终异丙基-β-D-硫代半乳糖苷（IPTG）浓度为 0.2 mM，诱导温度为 15°C，诱导时间为 16 h，确定了诱导表达条件。成功制备了具有低内毒素和无菌性的重组 PFAP-1 蛋白。重组 PFAP-1 蛋白在体外对耐甲氧西林金黄色葡萄球菌（MRSA）具有很强的抗菌活性，抑菌圈直径为 15.99±0.02mm。其最小抑菌浓度（MIC）和最小杀菌浓度（MBC）分别为 37.5μg/mL 和 150μg/mL，杀菌浓度下的溶血活性较低（11.21%）。重组 PFAP-1 蛋白显著抑制 MRSA 生物膜的形成并根除 MRSA 生物膜。它还对 1,1-二苯基-2-苦基肼基（DPPH）自由基具有很强的清除活性，半数最大抑制浓度（IC）为 40.83μg/mL。ACE2 抑制的 IC 为 5.66μg/mL。分子对接结果表明，PFAP-1 蛋白和 ACE2 蛋白的最佳对接分数为-267.78 kcal/mol，置信度为 0.913。稳定的结合复合物主要通过九组氢键、三组盐桥和许多疏水相互作用形成。总之，重组 PFAP-1 可以作为一种有前途的活性蛋白，用于食品、化妆品或医药。
